The transcription factor NF-kappaB plays a critical role in innate immune responses by augmenting the expression of many inducible, immediate response genes, including IL-1, IL-2, IL-2Ralpha, IL-6, IL-8, IL-12, TNF-alpha, beta-IFN, GM-CSF, COX-2, E-selectin and serum amyloid A protein. NF-kappaB acts as a critical coordinating element in the body's response to infection and injury, and thus is an important mediator of innate immunity. Recent studies demonstrating the importance of NF-kappaB activation by signaling through the Toll-receptors has highlighted their evolutionarily conserved role in host response to pathogens. It is also an essential element for the replication of different viruses, including HIV. NF-kappaB also serves as an important anti-apoptotic factor in many biological systems and its aberrant activation has been linked to the development of certain forms of cancer. Therefore understanding how NF-kappaB activity is regulated has great relevance for the development of novel therapies for the treatment of inflammatory diseases and cancer. The primary long-term objective of our research is to determine how the signals from the Toll-receptors, that induce the activity of NF-kappaB, are transduced and lead to the mounting of innate immune responses. In this application we propose to study the Toll-receptor signal transduction pathway by focusing on two novel adapter proteins, ECSIT and X, that we have recently isolated. We will use a variety of approaches, including generation and analysis of gene targeted animals, to study these molecules. We will also try to isolate potential adapter proteins that bind to the Toll-receptors and substitute for MyD88. We wish to delineate the sequence of events that are responsible for the induction of the IKB kinase (IKK) complex activity in response to signaling from Toll-receptors. In particular we wish to understand the role of NEMO/IKKgamma, the regulatory subunit of the IKK complex in the activation process. Finally we will study the possible mechanisms by which the IKK proteins facilitate the proteosomal degradation of phosphorylated and ubiquitinated IkappaB substrates.